ABSTRACT Apolipoprotein E (apoE) isoforms differ by single amino acids at residues 112 and 158 (apoE3: Cys112, Cys158; apoE3: Cys112, Arg158; apoE4: Arg112, Arg158). These small differences cause a profound change in the apoE?s structure and function. ApoE4 is associated with the highest risk of onset of Alzheimer?s disease (AD), whereas apoE2 is ?protective? or ?less risky? than apoE3. Defining the unique biological properties of apoE2 will provide insights into its protective roles and may suggest novel therapeutic approaches. This proposal builds upon our model of apoE involvement in AD. We posit that apoE is induced in injured/stressed neurons and undergoes neuron-specific proteolysis (apoE4>apoE3>apoE2) to generate a series of neurotoxic apoE fragments (12?29 kDa). The fragments escape the secretory pathway and enter the cytosol where they cause mitochondrial dysfunction and stimulate phosphorylation of tau. Susceptibility to proteolysis and fragment formation occurs secondary to domain interaction (Arg61 ionic interaction with Glu255) and displays an isoform hierarchy (apoE4>apoE3>apoE2). We hypothesize that unique features of apoE2 make it less of a risk for AD. We will test this hypothesis by comparing the effects of apoE2 expression in neurons in transgenic mice with those of our apoE3 and apoE4 mice and cultured neurons to determine how apoE2 modulates apoE-associated neuropathology and neurodegeneration. We will investigate the biochemical, histopathological and behavioral consequences of apoE2 expression. Quantitation and characterization of the apoE2 fragments in the brain and plasma will allow correlation of the fragments with the ?protective? or ?less risky? role of this isoform in neuropathology. Our data indicate that apoE2 may be a risk, but just much less. The structural features of apoE2, such as reduced susceptibility to domain interaction and the unique presence of Cys158, a residue known to significantly alter the conformation of the molecule, likely moderates the effects on neurons. Understanding the effects of these structural elements will provide insights into how apoE2 is protective. The impact of apoE2 on mitochondrial function will be studied. Numerous studies indicate that mitochondrial dysfunction is an early pathological event in AD. In cultured Neuro-2a cells and primary neurons from apoE transgenic mice we will determine the mechanisms of action of apoE2 on cellular mitochondrial activity and energetics and how apoE2 may alter/modulate mitochondrial function, as compared to apoE3 and apoE4. ApoE4 fragments interact with mitochondria and impair mitochondrial respiratory enzyme levels and activity. We will extend our studies to apoE2 and apoE2 fragments with mitochondrial activity. Alteration of mitochondrial proteins will be coupled with the latest proteomic techniques and elucidation of apoE2 interactomes to identify unique pathways involved in apoE2 modulation of metabolism. !